Like any other light, fluorescence enabling light may not be localized in a sample stronger than down to Abbe's diffraction limit at the wavelength of the fluorescence enabling light. Even under optimum optical conditions Abbe's diffraction limit is at half of the respective wavelength. With regard to the fluorescence light emitted out of the sample, Abbe's diffraction limit at the wavelength of the fluorescence light applies to assigning the fluorescence light to a certain area of the sample. Correspondingly, a measurement area, both in subjecting it to the fluorescence enabling light and in measuring the fluorescence light, may not be made smaller than the diffraction limit at the wavelength of the fluorescence enabling light and the fluorescence light, respectively. By means of fluorescence inhibiting light, the spatial resolution in imaging a structure or in tracking a particle in a sample may be increased beyond Abbe's diffraction limit. If a partial area of a measurement area which is subjected to the fluorescence inhibiting light covers the entire measurement area except of an intensity minimum of an intensity distribution of the fluorescence inhibiting light, the measured fluorescence light may only stem from this intensity minimum and may thus be assigned to the position of this intensity minimum in the sample. The dimensions of an intensity minimum of the fluorescence inhibiting light in which the intensity of the fluorescence inhibiting light is zero or at least so small that the fluorescence inhibiting light does not inhibit the emission of fluorescence light completely, whereas the intensity of fluorescence inhibiting light outside of this intensity minimum is so high that it completely inhibits the emission of fluorescence light by the fluorescence markers, may be reduced far below the diffraction limit at the wavelength of the fluorescence inhibiting light and thus also at the wavelengths of the fluorescence enabling light and the fluorescence light by means of increasing the light intensity of the fluorescence inhibiting light. As a result, a spatial resolution is achieved in imaging the structure of interest or in tracking a particle of interest in the sample, which is by a factor of at least 5, 10 or even more better than in common confocal scanning fluorescence light microscopy.
The fluorescence inhibiting light may inhibit the emission of fluorescence light by the fluorescence markers in different ways. In stimulated emission depletion (STED) fluorescence light microscopy, the fluorescence enabling light is fluorescence exciting light which transfers the fluorescence markers via an electronic transition into an excited state out of which the fluorescence markers return into their ground state under spontaneous emission of fluorescence light. The fluorescence inhibiting light depletes the excited state in that it stimulates the fluorescence marker for the emission of light at another wavelength than that one of the fluorescence light, which due its different wavelength can be separated from the fluorescence light which is spontaneously emitted out of the intensity minimum of the fluorescence inhibiting light.
In STED scanning fluorescence light microscopy, the fluorescence inhibiting light has to have a very high intensity outside the intensity minimum to de-excite again the fluorescence markers which have been excited for emission of fluorescence light by means of the fluorescence enabling light by means of stimulated emission before they spontaneously emit fluorescence light as the lifetime of the excited electronic state of the fluorescence markers is only short.
In REversible Saturable Optical Fluorescence Transitions (RESOLFT) scanning fluorescence light microscopy using switchable fluorophores, the fluorescence enabling light switches the fluorescence markers into a fluorescent state in which they are excitable for spontaneous emission of fluorescence light by additional fluorescence exciting light. By means of fluorescence inhibiting light, the switched on fluorescence markers are switched off again except of those in the area of the intensity minimum of the fluorescence inhibiting light. Fluorescence light whose emission is afterwards excited by additional fluorescence exciting light may then only stem from the area of the intensity minimum of the fluorescence inhibiting light.
In RESOLFT scanning fluorescence light microscopy, lower intensities of the fluorescence inhibiting light than in STED scanning fluorescence light microscopy are sufficient, because the switched on state of the fluorescence markers, even if not stable, has at least a longer lifetime than an electronic state out of which the fluorescence markers emit the fluorescence light. On the other hand, special switchable fluorescence markers are needed.
In Ground State Depletion (GSD) scanning fluorescence light microscopy, fluorescence markers are transferred by the fluorescence inhibiting light via an electronic transition out of their ground state into a dark state in which they are not excitable for the spontaneous emission of fluorescence light when subjecting the sample to fluorescence excitation light as fluorescence enabling light. Fluorescence light measured afterwards may also here only stem from the intensity minimum of the fluorescence inhibiting light.
In GSD scanning fluorescence light microscopy, it is difficult to transfer the fluorescence markers completely into their dark state on the one hand, and to quickly return them back into their ground state, when a neighboring measurement area of the sample is to measured, on the other hand.
V. Westphal and S. W. Hell: Nanoscale Resolution in the Focal Plane of an Optical Microscope, PRL 94, 143903 (2005) disclose a method of high-resolution imaging a structure of a two-dimensional sample, the structure being marked with fluorescence markers, and the method belonging to STED scanning fluorescence light microscopy. In addition to excitation light having a central intensity maximum, fluorescence inhibiting light is provided with a line-shaped intensity minimum. In a comparison example, two partial intensity distributions of the fluorescence inhibiting light which each comprise a line-shaped intensity minimum are superimposed with orthogonal lines to define a point-shaped intensity minimum. For forming the two partial intensity distributions, the fluorescence inhibiting light is split up into two partial beams. With a fixed light power of the fluorescence inhibiting light, a maximum spatial resolution is achieved when using the fluorescence inhibiting light with the line-shaped intensity minimum. This maximum spatial resolution is achieved in a spatial direction orthogonal to the line-shaped intensity minimum. In the direction of the line-shaped intensity minimum, however, the spatial resolution is only that one of a confocal scanning fluorescence light microscope. When the spatial resolution is increased in both spatial directions of the two-dimensional sample by means of the point-shaped intensity minimum using the same light power of the fluorescence inhibiting light, the spatial resolution is considerably smaller than the maximum spatial resolution achieved by means of a line-shaped intensity minimum of the fluorescence inhibiting light.
US patent application publication US 2012/0104279 A1 discloses a method of high-resolution imaging a structure of a sample marked with fluorescence markers, which, in one embodiment, belongs to STED scanning fluorescence light microscopy. This known method may, however, also be executed according to GSD or RESOLFT scanning fluorescence light microscopy. Fluorescence inhibiting light is provided with a donut-shaped intensity distribution around a point-shaped intensity minimum. The field vector of the electrical field in the donut may rotate or have a fixed orientation to either inhibit the fluorescence of all fluorescence markers in the area of the donut independently on their dipole orientation, or to purposefully only inhibit fluorescence of those fluorescence markers whose dipoles are orthogonal to the fixed orientation of the field vector.
US patent application publication US 2007/0206278 A1 discloses a method of high-resolution imaging a structure of a two-dimensional sample marked with fluorescence markers, which may be implemented as a method of either RESOLFT or GSD scanning fluorescence light microscopy. In the RESOLFT embodiment of the known method, the fluorescence markers in the sample are at first transferred into a state in which they are able to fluoresce by means of fluorescence enabling light in a line-shaped measurement area. In the GSD embodiment of the known method, it is waited until the fluorescence markers have returned into their ground state in which they are able to fluoresce. In both embodiments, the fluorescence markers in the line-shaped measurement area are then subjected to an intensity distribution of fluorescence inhibiting light which transfers the fluorescence markers, except of those fluorescence markers which are located in a line-shaped intensity minimum of the fluorescence inhibiting light in the center of the line-shaped measurement area, out of their state in which they are able to fluoresce into a dark state. Afterwards, the fluorescence markers in the line-shaped measurement area are subjected to fluorescence excitation light. The fluorescence light which is then emitted out of the line-shaped measurement area of the sample is measured with a line detector, i.e. with a detector spatially resolving in a direction along the line. The steps described here are repeated for a plurality of measurement areas to scan the sample with the line-shaped intensity minimum of the fluorescence inhibiting light. This scanning may be sequently executed with different orientations of the line-shaped intensity minimum of the fluorescence inhibiting light, and from the plurality of images of the sample obtained in this way, an overall image may be calculated mathematically which has an increased spatial resolution in several spatial directions. The known method shall speed up scanning of the sample as compared to a pointwise scan. Very high intensities of the fluorescence inhibiting light which are needed for STED scanning fluorescence light microscopy may not be realized over an extended line-shaped measurement area in a suitable way, because the light power of the fluorescence inhibiting light is distributed over a too large sample volume.
US patent application publication US 2013/0176574 A1 discloses a method and a scanning fluorescence light microscope for multi-dimensional high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers. Here, the sample is only subjected to fluorescence excitation light. The fluorescence excitation light is focused to a measurement area with diffraction-limited dimensions, and fluorescence light emitted out of the measurement area is measured with a point detector. For increasing the spatial resolution, the phase fronts of the fluorescence excitation light are modulated prior to focusing the fluorescence excitation light within the sample in such a way that different interference patterns are formed in the measurement area. These interference patterns may include interference patterns having line-shaped intensity minima oriented at different angles. The sample is completely scanned with each of these interference patterns, and the measurement values of the fluorescence light belonging to the different interference patterns are mathematically evaluated together to obtain an image with a spatial resolution increased better than the size of the measurement area. Alternatively, all different interference patterns may be adjusted successively at each point of the sample, before the next point is measured in scanning the sample with the measurement area. In this embodiment, the fluorescence light emitted out of the measurement area is also separately registered for each of the different interference patterns.
German patent application publication DE 10 2011 055 367 A1 (corresponding to U.S. Pat. No. 9,291,562 B2) discloses a method and a scanning fluorescence light microscope for tracking a movement of a particle in a sample, the particle being marked with a fluorescence marker. Fluorescence excitation light having an intensity distribution with a spatially limited minimum is directed onto the sample, and the minimum is guided to track the particle moving within the sample in that the intensity distribution of the fluorescence excitation light is shifted with regard to the sample in such a way that a rate of photons of the fluorescence light emitted by the particle remains minimal. The rate of photons of the fluorescence light emitted by the particle only remains minimal if the particle remains in the minimum of the intensity distribution of the fluorescence excitation light. Different phase relations between light beams from which the intensity distribution of the fluorescence excitation light is generated by means of interference may successively result in line-shaped or plane-shaped minima oriented in different directions. These line-shaped or plane-shaped minima oriented in different directions are called rotating stripes and have a point or a line as their spatial intersection. With rapidly switching over between such different phase relations and with keeping the rate of photons minimal for each of these phase relations individually or over the entirety of the different phase relations, the movement of the particle in the sample can be tracked in all three dimensions.
In Image Scanning Microscopy (ISM), a structure in a sample which is marked with fluorescence markers is scanned with a diffraction-limited measurement area into which fluorescence excitation light is focused as fluorescence enabling light in a same way as in common confocal scanning fluorescence light microscopy. In contrary to common confocal scanning fluorescence light microscopy, for each position of the measurement area, the fluorescence light emitted from the sample out of the measurement area is registered not just confocally but with a sensor array, the intensity distribution of the fluorescence light over the sensor array being registered. Due to the diffraction limit, the diffraction-limited measurement area may not be resolved spatially by means of the sensor array. Nevertheless, additional information with regard to the position of the fluorescence light emitting fluorescence markers is obtained. At first, four- or five-dimensional data sets are produced in which two or three dimensions correspond to the position of the measurement area in the sample and two further dimensions correspond to the coordinates within the sensor array at which the fluorescence light has been registered for the respective position of the measurement area. From these data sets, a final image having an increased spatial resolution may be calculated. The maximum spatial resolution which is achievable in this way corresponds to the spatial resolution achievable in Structured Illumination Microscopy (SIM), and it is by a factor of 2 better than the spatial resolution in common confocal scanning fluorescence light microscopy. A method of ISM and a corresponding scanning fluorescence light microscope are, for example, described in European patent application publication EP 2 317 362 A1 (corresponding to U.S. Pat. No. 8,705,172 B2). In more detail, ISM is described by Claus B. Müller and Jörg Enderlein: Image Scanning Microscopy. Physical Review Letters, Vol. 104, 198101 (2010). The mathematical basics of ISM have already been disclosed by C. J. R. Sheppard: Super-resolution in confocal imaging. Optik, 80 No. 2 (1988) 53-54.
A direct optical realization of the evaluation which in ISM is otherwise executed mathematically is described by Stephan Roth, Colin J R Sheppard, Kai Wicker and Rainer Heintzmann: Optical photon reassignment microscopy (OPRA); Optical Nanoscopy 2013, 2:5.
There still is a need of a method of multi-dimensional high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, which achieves a desired spatial resolution in all of the multi dimensions at a lower light power of the fluorescence inhibiting light. Further, a corresponding method of multi-dimensional high-resolution imaging a path of a particle in a sample, the particle being marked with a fluorescence marker, and scanning fluorescence light microscopes for carrying out these methods are needed.